AIDS is one of the leading causes of death in the developing world, its spread reaching pandemic proportions. However, eradication of HIV-1 is far from being accomplished. Currently, the highly active anti-retroviral therapy (HAART) is the only efficacious treatment to reduce progression and spread of AIDS, although its long-term use is associated with drawbacks and limitations such as adherence to a complex dosing regimen, side effect toxicity and elevated cost (Richman et al., 2001). The great intra- and inter-subtype genetic and antigenic variability of HIV-1, stemming from the high mutation rate of its genome, together with inadequate compliance, is responsible for resistance to HAART drugs, as well as for the repeated failure in developing a multiple clades-based preventive vaccine (Ho et al., 2002). On this basis, development of alternative and/or additional therapeutic strategies against AIDS are mandatory.
Many years of pre-clinical investigation have shown that the HIV-1 life cycle can be interfered with at many levels, and proved at least the concept of anti-HIV gene therapy (Buchschacher et al., 2001). Hematopoietic stem cells (HSCs), T-cell precursors or T lymphocytes can be genetically modified with, for example, genes encoding ribozymes, decoys, antisense and small interfering RNA (siRNA) molecules directed against viral and cellular genes (Buchschacher et al., 2001; Jacque et al., 2002; Novina et al., 2002; Lee et al., 2002; Coburn et al., 2002; Qin et al., 2003), or proteins such as intrakines, toxins and single chain antibodies. However, early clinical trials with T lymphocytes transduced with retroviral vectors expressing transdominant mutants of viral proteins or anti-HIV-1 rybozimes have been disappointing (Woffendin et al., 1996; Ranga et al., 1998; Wong-Staal et al., 1998) mainly due to low gene transfer efficiency, insufficient engraftment and short in vivo persistence of the genetically modified T cells. Most of the pre-clinical and clinical studies carried out so far have been based on the use of retroviral vectors derived from the Moloney murine leukemia virus (MLV) to transduce HSCs or T-cells. However, MLV-derived vectors have shown major limitations for clinical applications, such as poor efficiency in transducing non-dividing HSCs and T-cells, insufficient expression of potentially therapeutic anti-HIV products, and propensity to induce neoplasia by insertional activation of oncogenes (Baum et al., 2003).
Among the possible targets of anti-HIV gene therapy is the product of the viral infectivity factor (vif) gene. vif is one of the 4 accessory genes of HIV-1, expressed at a late phase during virus replication in a Rev-dependent manner (Cullen et al., 1998; Frankel et al., 1998). The Vif protein is required for high viral infectivity in the so-called ‘non-permissive’ cells, which include the natural targets of HIV-1 (T-cells and macrophages) and some T-cell lines, for example, CEM, H9, and HUT 78 (Fisher et al., 1987; Fouchier et al., 1996; Gabuzda et al., 1992; Sheehy et al., 2002; Simon et al., 1996; von Schedler et al., 1993). This requirement depends on the ability of Vif to counteract the action of the recently identified CEM15/APOBEC-3G protein (Sheehy et al., 2002) which confers innate immunity to HIV-1. Thus, disabling, or interfering with, the function of Vif could represent an alternative anti-HIV-1 therapeutic approach.
F12-vif is a natural mutant of vif, carrying 15 unique amino acid substitutions, originally discovered in the F12 non-producer variant of HIV-1 (Federico et al., 1989; Carlini et al., 1992; Carlini et al., 1996). The F12 non-producer HIV induces a block in the replication of superinfecting HIV and F12-Vif may play a role in the reduced infectivity of this producer. However, there is a need to provide further vif mutants with anti-HIV activity, and to provide effective delivery systems for these mutants. The present invention seeks to overcome these problems.